Structural and Functional Plasticity of the Staphylococcus aureus Virulence-Associated Amyloid Peptide PSMα1

Abstract

Phenol-soluble modulin α1 (PSMα1) is a cytolytic peptide secreted by Staphylococcus aureus that contributes to host-cell damage and biofilm stability, yet the relationship between its assembly behavior and function remains incompletely understood. Here, we combine cellular assays, molecular spectroscopy, and high-resolution structural approaches to elucidate how environmental conditions govern PSMα1 activity and supramolecular organization. Live-cell imaging and cytotoxicity assays show that PSMα1 accumulates at the plasma membrane of human cells prior to membrane permeabilization, linking membrane association to cytotoxic outcomes. This process is strongly attenuated by epigallocatechin gallate (EGCG). Cryogenic electron microscopy (cryo-EM) reveals two polymorphic canonical amyloid fibril architectures that share a conserved hydrophobic core and protofilament interface. In parallel, we identify pH as a key determinant of PSMα1 assembly pathways, driving a bifurcation between cross-β amyloid fibrils at extreme acidic and alkaline conditions and heterogeneous, long-lived, thermally stable α-helical nanotubular assemblies at acidic, near-neutral, and slightly alkaline conditions, which act as transient intermediates under highly acidic conditions. Together, these findings demonstrate that PSMα1 is not a single amyloid structure but a condition-dependent structural system in which environmental cues dictate assembly, membrane interaction, and cytotoxic function. This work provides a framework for understanding how polymorphic assembly of bacterial virulence peptides interfaces with host-cell interactions and suggests new avenues for targeting PSM-mediated pathogenicity. Statement of significance Staphylococcus aureus causes severe infections and uses the peptide PSMα1 to damage host cells and strengthen protective biofilms. Like many disease-associated proteins, PSMα1 self-assembles into amyloid fibrils, though their role in virulence remains unclear. We show that PSMα1 does not adopt a single architecture. Instead, environmental changes, such as those at infection sites, drive the peptide into distinct assemblies, including cross-β amyloid fibrils and unexpectedly stable nanotubes with α-helical features. Live-cell imaging shows PSMα1 accumulates at the plasma membrane before cell death, and that epigallocatechin gallate reduces membrane association and toxicity. These findings show that bacterial virulence can be regulated through environmentally controlled transitions between protein assemblies, identifying membrane accumulation as a promising anti-virulence target.